1. Field of the Invention
The present invention relates to the use of selenone compounds as anti-cancer agents, and to methods of making these compounds. Particularly, the present invention relates to the use of selenone compounds as anti-cancer alkylating agents against cell lines which display resistance to conventional anti-cancer alkylating agents.
2. Background
Classical alkylating agents may be defined as compounds that in protic media undergo aliphatic nucleophilic substitution reactions at saturated, sp.sup.3 -carbon electrophilic centers bearing an acidic leaving group. Discovery of the antitumor properties of mechlorethamine hydrochloride (nitrogen mustard) led to the synthesis of thousands of (2-haloethyl)imonium, aziridine, (2-haloethyl)sulfonium, and oxygen analogues by the early 1960's and to the development of alkylating agents as an established class of cancer chemotherapy agents. Platinating agents are also an established class of cancer chemotherapy agents. The ligand substitution reactions of platinating agents are essentially unimolecular in overall kinetics, and follow a very similar nucleophilic reactivity order. That is, in protic media (such as the biological intracellular situation), the second-order rate constants for nucleophilic substitution are in decreasing order: S.sub.2 O.sub.3.sup.2- &gt;RS--&gt;R.sub.2 N.about.N.sub.3 --&gt;CH.sub.3 COO--.about.Cl--&gt;NO.sub.3 --&gt;H.sub.2 O. The range of values over this entire sequence is typically one-million fold or more. For classical alkylating agents and platinating agents, the anionoid electron pair of nitrogen is typically hundreds to tens of thousands times more reactive than hydrolysis with water.
The properties of nucleophilicity and electivity can be quantitatively expressed as n and s constants in the Swain-Scott linear free energy relationship. It has been shown that s and n constants of biological alkylating agents and nucleophiles, respectively, can be readily determined with high precision, using 4-(4-nitrobenzyl)pyridine (NBP) as a competitive substrate for alkyl product formation (Spears, C. P. Mol. Pharmacl.19:496-504,1981; Barbin,A.,Bereziat, A., O'Neill, I. K., and Bartsch, H. Chem.-Biol. Interact,73:261-277, 1990; Kang, S. I., and Spears,C. P. J. Med. Chem. 33:1544-1547, 1990). Broad-spectrum antitumor alkylating agents show uniformly high s constants Spears, C. P. Mol.Pharmacol. 19:496-504, 1981; in contrast, mutagenically efficient alkylating agents show low values (Peterson,A. R., Landolph, J. R., Peterson, H., Spears, C. P., and Heidelberger, C. Cancer Res. 41:3095-3099, 1981; Barbin, A., and Bartsch, H. Mut. Res.215:95-106, 1989).
Extensive literature supports the hypothesis that alkylation at N7-guanine in DNA by ethyleneimines and platinating agents mediates the major cytotoxic effects of these agents. The basis for this high sensitivity for N7-G adduct formation (Brookes,P., and Lawley,P. D. Biochem. J.78:3923-3928, 1961) is the high nucleophilicity at N7-G (Pullman, A., and Pullman, B. Quart. Rev. Biophys. 14:289-380,1981) plus the unusually high selectivities of ethyleneimines and platinating agents for electron-rich nucleophiles (Spears,C. P. Mol. Pharmacol.19:496-504, 1981; Pearson, R. G., Sobel, H.., and Songstad, J. J. Am. Chem. Soc. 90:319-326, 1968; Ibne-Rasa, K. M. J. Chem. Educ. 44:89-94, 1967. Kreuger,J. H., Sudburg, B. A., and Blanchet, P. F. J. Am. Chem. Soc.96:5733-5736, 1974; Edwards, J. O. Inorganic Reaction Mechanisms. W. A. Benjamin, N.Y., 1965, pp. 51-89). Maxam-Gilbert DNA sequence analysis has shown that marked preferential attack occurs by ethyleneimines, generally at runs of guanines (Mattes, W. B., Hartley, J. A., and Kohn, K. W. Nucl. Acids Res. 14:2971-2987, 1986). These ethyleneimines include mechlorethamine, chlorambucil, and L-phenylalanine mustard. Studies have suggested 50-fold increases in reaction rates for native DNA over free deoxyguanosine (Price, C. C., Gaucher, M., Koneru, P., et al. Biochim. Biophys. Acta 166:327-359, 1968). This increase has been tied to the enhanced electrostatic potential at N7 by the presence of flanking guanines, and is not an effect of cross-linking (Gralla, J. D., Sasse-Dwight, S., and Poljak, L. G. Cancer Res. 47:5092-5096, 1987). Runs of guanines, such as the regulatory sequence, GGGCGG in SV40 DNA, may have an important role in oncogene expression. Recently, evidence has been presented that guanine-rich sequence preferences for alkylation by ethyleneimines occur in coding regions of c-myc and N-myc oncogenes. (Gralla, J. D., Sasse-Dwight, S., and Poljak, L. G. Cancer Res. 47:5092-5096, 1987; Futscher, B. W., and Erickson, L. C. Proc. Am. Assoc. Cancer Res. 29:468, 1988; Kallama, S., and Hemminki, K. Chem.-Biol. Interact. 57:85-96, 1986). Conceptual advantages of the use of platinating agents over classical alkylating agents include the narrow cross-linking distance of the biologically effective cis-compounds (3.4 .ANG.), which is about one-half that of ethyleneimines. The other major difference is the occurrence of extremely high, calculable s constants of platinating agents. (Pearson, R. G., Sobel, H., and Songstad, J. J. Am. Chem. Soc. 90:319-326, 1968; Ibne-Rasa, K. M. J. Chem. Educ. 44:89-94, 1967. Kreuger, J. H., Sudburg, B. A., and Blanchet, P. F. J. Chem. Soc. 96:5733-5736, 1974; Edwards, J. O. Inorganic Reaction Mechanisms. W. A. Benjamin, N.Y., 1965, pp. 51-89). These differences could explain the ascendancy of platinating agents over ethyleneimines in the treatment of human ovarian cancer, germ cell neoplasms, and head and neck cancer, (DeVita, V. T., Jr., Hellman, S., and Rosenberg, S. A. (eds) Cancer. Principles and Practice of Oncology, 3rd Edition, 1989, pp. 495-503, 584-590, 657-705, 1084-1098, 1177-1196) despite the heavy metal toxicities.
Systematic chemical modifications of nitrosoureas and triazenes in the 1960s led to the discovery of 2-chloroethylating antitumor agents. These agents have desirably short cross-linking distances similar to those of platinating agents. This class of cross-linking agents includes BCNU (carmustine), CCNU (lomustine), and MeCCNU (semustine). (Schabel, F. M., Jr. Cancer Treat. Rep. 60:665-6, 1976). They are highly active in vivo against a broad range of murine neoplasms, but have demonstrated relatively narrow clinical activity. Clomesone and Cyclodisone, derivatives of sulfonates, are the most recent examples of bifunctional 2-chloroethyl derivatives (Gibson, N. W. Cancer Res. 49:154-157, 1989), currently under phase I clinical development.
In contrast to classical alkylating and platinating agents, the nitrosourea 2-chloroethylating agents react with DNA at a wide variety of nucleophilic sites (Barbin, A., and Bartsch, H. Mut. Res.215:95-106, 1989, Barbin, A., Bereziat, A., O'Neill, I. K., and Bartsch, H. Chem.-Biol. Interact. 73:261-277, 1990, Tong, W. P., Kohn, K. W., and Ludlum, D. B. Cancer Res. 42:4460-4464, 1982; Bartsch, H., Terracini, B., Malaveille, C., et al. Mut. Res. 110:181-219, 1983. Newbold, R. F., Warren, W., Medcalf, A. C. S., and Amos, J. Nature 283:596-599, 1980). In particular, significant product formation at the weakly nucleophilic O6-G site occurs. After 2-chloroethyl group transfer, cross-link formation may be intra- or inter-molecular at either the O6- or N7-G position. This further increases the plethora of products. A major chemical basis for the diverse product spread is the fact that nitrosoureas possess inherently low nucleophilic selectivities (Spears, C. P. Mol.Pharmacol. 19:496-504 1981; Peterson, A. R., Landolph, J. R., Peterson, H., Spears, C. P., and Heidelberger, C. Cancer Res. 41:3095-3099, 1981; Barbin, A., and Bartsch, H. Mut. Res. 215:95-106, 1989; Bartsch, H., Terracini, B., Malaveille, C., et al Mut.Res.110:181-219, 1983). On the other hand, it has been shown (by the inventors) that Clomesone, which may be therapeutically superior to nitrosourea 2-chloroethylating agents, has relatively high nucleophilic selectivity approaching that of chlorambucil (Kang, S. I., and Spears, C. P. J. Med. Chem. 33:1544-1547, 1990).
Problems exist with the use of prior alkylating agents, platinating agents and nitrosoureas as anti-cancer agents. Specifically, some cell lines demonstrate resistance to conventional alkylating agents, platinating agents and nitrosoureas.
As noted above, reaction at the O6-G site is a major mechanism of the cytotoxicity of nitrosoureas. A specific DNA repair enzyme, O6-G-alkyltransferase, can be induced to mediate resistance to this mechanism. Cells with increased levels of this enzyme are termed "MER(+)". Increased levels of this enzyme are associated with marked resistance to 2-chloroethylating agent cytotoxicity (Gibson, N. W. Cancer Res.49:154-157, 1989; Dolan, M. E., Pegg, A. E., Hora, N. K., and Erickson, L. C. Cancer Res. 48:3603-3606, 1988; Dolan, M. E., Norbeck, L., Clyde, C., Hora, N. K., et al. Carcinogenesis 10:1613-1619, 1989; A. E. Pegg, Cancer Res. 50:6119-6129, 1990). The occurrence of such enzymes in human cancers has been proposed as a mechanism for the surprisingly limited clinical antitumor activity of nitrosoureas when compared to their effectiveness in animal tumors. This resistance is a major problem with the clinical use of nitrosoureas (A. E. Pegg, Cancer Res. 50:6119-6129, 1990).
In contrast, the highly selective ethyleneimine and platinating agents are associated with, and induce drug resistance mediated by, a variety of intracellular thiol mechanisms. One such mechanism is elevation of intracellular reduced glutathione (GSH) to low millimolar concentrations. This is a common occurrence with high s constant nitrogen mustards and cis-diaminodichloroplatin (cisplatin or CDDP) (Dolan, M. E., Norbeck, L., Clyde, C., Hora, N. K., et al. Carcinogenesis 10:1613-1619, 1989; Ball, C. R., Connors, T. A., Double, J. A. Ujhazy, V., and Whisson, M. E. Int. J. Cancer 1:319-327, 1966; Dorr, R. T. Biochem. Biophys. Res. Commun.144:47-52, 1987; Kramer, R. A., Greene, K., Ahmed, S., and Vistica, D. T. Cancer Res. 47:1593-1597, 1987; Hamilton, T. C., Masuda, H., and Ozols, R. F. In: Resistance to Antineoplastic Drugs (Kessel, D., ed.), CRC Press, Boca Raton, Fla., 1989, pp.49-61). These alkylating agents with high s constants preferentially alkylate the sulfur atom of GSH, instead of forming the cytotoxic N7-G adduct. Drug resistance of this type may be overcome by various interventions, such as inhibition of glutathione reductase by buthionine sulfoxamine (Dorr, R. T. Biochem.Biophys.Res. Commun. 144:47-52, 1987; Kramer, R. A., Greene, K., Ahmed, S., and Vistica, D. T. Cancer Res. 47:1593-1597, 1987), which markedly depletes the levels of GSH.
Glutathione mechanisms and elevated levels of intracellular thiols can also strongly contribute to the resistance of cancer cells to doxorubicin and other antibiotic antitumor agents. Such resistance can also be reversed by thiol depletion treatment (buthionine sulfoxamine as described above) (Hamilton, T. C., Masuda, H., and Ozols, R. F. In: Resistance to Antineoplastic Drugs (Kessel, D., ed.), CRC Press, Boca Raton, Fla., 1989, pp.49-61).
Both enzymic and non-enzymic mechanisms for thiol-mediated drug resistance are known to occur in the presence of agents with high s constants. Thus, glutathione transferase activity can substantially contribute to the resistance of tumors to ethyleneimines and aziridines (Robson, C. N., Lewis, A. D., Wolf, C. R., et al. Cancer Res.47:6022-6027, 1987). In the case of cisplatin, metallotheinine, a thiol-rich peptide, can mediate resistance (Naganuma, A., Sakoh, M., and Imura, N. Cancer Res. 47:983-987, 1987).
In contrast to the case of high s constant alkylating agents, alkylating agents with low s-constants (such as the nitrosoureas which display only modestly greater reactivities for sulfur over nitrogen nucleophiles) rarely appear to suffer from resistance on the basis of a thiol mechanism (Aida, T., and Bodell, W. J. Cancer Res.47:1361-1366, 1987; Bedford, P., Walker, M. C., Sharma, H. L., et al. Chem. Biol. Interact. 61:1-15, 1987). As noted above, though, they suffer from MER(+) drug resistance.
Thus, it would be desirable to obtain cytotoxic compounds which possess high selectivity for N7-guanine yet avoid thiol-mediated mechanisms of resistance. High selectivity avoids MER(+)-mediated resistance. The present invention provides selenone compounds that satisfy these requirements.
Although the synthesis of bis(2-chloroethyl) selenide was first described in 1920, the antitumor potential of 2-haloethyl selenium compounds, as selenides, was not known until 1987. (Kang S. I. and Spears C. P., J. Med. Chem. 30:597-602, 1987). This was surprising, given the antitumor activities of inorganic selenium (Poirier, K. A., and Milner, J. A. J.Nutr. 113:2147-2154, 1983), the promising antitumor activity of selenium antimetabolite analogs, (Melvin, J. B., Haight, T. H., and Leduc, E. H. Cancer Res. 44:2794-2798, 1984), (Safayhi, H., Tiegs, G., and Wendel, A. Biochem. Pharmacol. 34:2691-2694, 1985), (Muller, A., Cadenas, E., Graf, P., and Sies, H. Biochem. Pharmacol. 33:3235-3239, 1984), the anticarcinogenic effect of dietary selenium (Shamberger, R. Mut. Res. 154: 29-48, 1985), which is an essential trace element (Buell, D. N. Semin.Oncol.10:311-321, 1983), the important role of selenium in glutathione metabolism (Chung, A. S., and Maines, M. D. Biochem. Pharmacol. 30:3217-3223, 1981), and the requirement of some cancer cell lines in vitro for selenium as a nutrient (Carney, D. N., and De Leiji, L. Semin. Oncol. 15: 199-214, 1988).
The inventor's interest in 2-haloethyl selenium compounds derived from theoretical considerations of alkylating agent nucleophilic selectivity. High nucleophilic selectivity in an alkylating agent, as represented by the s constant of Swain and Scott, should display increased alkylation of N7-G in DNA and of other moderately strong intracellular nucleophilic sites.
A structural feature of alkylating agents which favors increased nucleophilic selectivity is high polarizability in the leaving group and of other atoms located near the reaction center (Bunnett, J. F. Annu.Rev.Phys.chem.14:271-290, 1963; Edwards, J. O., and Pearson, R. G. J. Am. Chem.Soc.84:16-24, 1962; Edwards, J. O. J. Am. Chem. Soc. 76:1540-, 1954; Edwards, J. O. Inorganic Reaction Mechanisms. W. A. Benjamin, N.Y., 1965, pp.51-89). Since nitrogen and sulfur atoms act in part as leaving groups in the final alkylation reactions of ethyleneimonium (Price, C. C., Gaucher, G. M., Koneru, P., et al. Ann. N.Y. Acad. Sci 163:593-600, 1969) and ethylenesulfonium ions, (Ingold, C. K. Structure and Mechanism in Organic Chemistry. Cornell Univ. Press, Ithaca, N.Y., 1953, pp. 384-385) selenium analogues were logical candidates for study because of the large, "soft" character of selenium (Ho, T.-L. Chem.Rev.75:1-20, 1975). In a report of s constant determination of model and clinical alkylating agents, the presence of an aromatic nucleus at the ethyleneimonium nitrogen also appeared to increase nucleophilic selectivity (Spears, C. P. Mol.Pharmacol.19:496-504, 1981). A comparison of reactivities, nucleophilic selectivities, and cytotoxicities of monofunctional 2-haloethylarylselenides was done with bifunctional aliphatic analogues (Kang, S. I., and Spears, C. P. J.Med.Chem. 30:597-602, 1987), (Kang, S. I., and Spears, C. P. J.Pharm.Sci. 79:57-62, 1990). It was found that 2-haloethyl selenides show a surprisingly wide range of values in reactivities, selectivities, and cytoxicities. Useful correlations between these values and Hammett constants were demonstrated. Some of the selenides were among the most reactive alkylating agents that have ever been described (making these potentially useful by topical application, intraarterial infusion, or intracavitary administration).
However, selenide alkylating agents suffer a number of drawbacks which appear to limit their clinical potential. The extremely high reactivities of the 2-chloroethyl selenides clearly is a problem for systemic intravenous therapy. The requirement for two selenium alkylating centers plus their generally poor aqueous solubilities discourage further preclinical development.
However, the selenides are useful as the immediate precursors for synthesis of 2-chloroethyl aryl selenones. The selenones of the present invention possess desirable solubilities and reactivities, short cross-linking distances, and show unusual and striking selectivities for N7-G type nucleophiles. The synthesis of monofunctional 2-chloroethyl arylselenides is carried out using diselenide or selenocyanate intermediates (Kang, S. I., and Spears, C. P. J. Med. Chem. 30:597-602, 1987). variation in alkyl chain length, halide leaving group, and bifunctionality (in terms of Se centers) can be achieved using hydroxyalkyl selenocyanate intermediates (Kang, S. I., and Spears, C. P. Synthesis 133-135, 1988; Kang, S. I., and Spears, C. P. J.Pharm.Sci.79:57-62, 1990).
Reich (Organoselenium Chemistry, J. Wiley & Sons, N.Y. 1987, p. 258) has commented that the chemistry of selenones has been little studied compared to sulfur analogues because harsh conditions for oxidation are required for synthesis, and because of the instabilities of selenoxides and selenones. Simple dialkyl selenones were synthesized from the corresponding dialkyl selenoxides by ozonation in a 1968 report (Paetzold, R., and Bochman, G. Z. Anorg. Allgem. Chem. 360:293-, 1968). Perhydrol treatment of the selenide was used later, to prepare selenacyclohexane-1,1-dioxide (Lambert, J. B., Mixan, C. E., and Johnson, D. H. J. Am. Chem. Soc. 95:4634-4639,1973). Peracetic acid and hydrogen peroxide have been useful to oxidize substituted diphenylselenides (or selenoxides) to the selenone analogs (Rebane, E. Chem.Scripta 5:65-, 1974; Bergman, J., Engman, L., and Siden, J. In: The Chemistry of Organic Selenium and Tellurium Compounds, Vol. 1. S. Patai and Z. Rappoport, eds., John Wiley and Sons, N.Y., 1986, pp. 546-558). Potassium permanganate has also been reported as useful for preparation of diphenyl selenones, but was unsuccessful for dimethylselenone from the selenide. A method which results in low yields for preparation of diphenyl selenones is direct selenonation of aromatic precursors with selenium trioxide, SeO.sub.3. (Bergman, J., Engman, L., and Siden, J. In: The Chemistry of Organic Selenium and Tellurium Compounds. Vol. 1. S. Patai and Z. Rappoport, eds., John Wiley and Sons, N.Y., 1986, pp. 546-558).
The history of organoselenones (R.sub.1 Se(O.sub.2)R.sub.2) as a class has been reviewed. (Bergman, J., Engman, L., and Siden, J. In: The Chemistry of Organic Selenium and Tellurium Compounds. Vol. 1. (S. Patai and Z. Rappoport, eds.), John Wiley and Sons, N.Y., 1986, pp. 546-558.; Paulmier, C. Selenium Reagents and Intermediates in Organic Synthesis. Pergamon Press, N.Y., 1986, pp. 153-161; Reich, H. J. In: Proceedings of the Fourth International Conference on the Organic Chemistry of Selenium and Tellurium (F. J. Berry and W. R. McWhinnie, eds.), 1983, pp. 268-272. To date, no selenone compounds other than those of the present invention have been proposed as antitumor agents.
A variety of reactions are known to take place using organoselenones. Alkyl selenones of the type PhSe(O.sub.2)CH.sub.2 CHRCH.sub.3, where R.dbd.CH.sub.3 or Ph, undergo syn-elimination reactions with heating (80.degree.-100.degree. C.) to produce olefins as well as products of reactions with benzeneseleninic and/or selenenic acid. The later side products are prevented from forming by the addition of 2-methoxypropene as a PhSeOH trap (Reich, H. J. In: Organoselenium Chemistry D. Liotta, ed., J. Wiley and Sons, Wiley Interscience, N.Y., 1987, p. 258). This type of selenone leaving group behavior has been used for synthesis of oxetanes, by treatment of selenones with NaOH in aqueous methanol (Shimizu, M., and Kuwajima, I. J. Org. Chem.45:4063-4065, 1980).
The alpha-protons of selenones are acidic, 2 pK.sub.a units more acidic than corresponding sulfones, which allows reaction by a .alpha.-selenonyl carbanion intermediate, as a second pathway, which is however infrequently observed (Reich, H. J. In: Organoselenium Chemistry D. Liotta, ed., J. Wiley and Sons Wiley Interscience, N.Y., 1987, p. 258).
A third leaving group reaction of organoselenones is that of facile SN2 displacement, originally mentioned by Reich who suggested that such behavior could make PhSeO.sub.2 CH.sub.3 a biological alkylating agent (Reich, H. J. In: Organoselenium Chemistry (D. Liotta, ed.), J. Wiley and Sons (Wiley Interscience), N.Y., 1987, p. 258). Although PhSeO.sub.2 CH.sub.3 was "three times as reactive as methyl iodide" (in methanol, 35.degree. C.) in methylation of dimethylsulfide (to produce (CH.sub.3).sub.3 S+ and PhSe(.dbd.O)O), no rate data was provided. In addition, although nucleophiles such as alkoxides, cyanide, and amines were also mentioned to undergo similar SN2 displacements with PhSeO.sub.2 CH.sub.3, no information on the nucleophilic reactivity order was provided.
Other evidence that organoselenones can undergo SN2 nucleophilic displacement (similar to reactions of the reactive intermediates of classical alkylating agents) has been very limited. ArSeO.sub.2 CF.sub.3 was observed to undergo haloform-type decomposition under mildly basic conditions (OH--) to HCF.sub.3 and ArSe(.dbd.O).sub.2 O. The early preparation (J. Loevnich, et al., Ber. Dtsch Chem. Ges., Vol. 62, pp. 2856-2865, 1929) of 2-NaphthSeO.sub.2 CH.sub.3 from 2-NaphthSeO.sub.2 Na and CH.sub.3 I is also probably an SN2-type reaction (in reverse) showing the high nucleophilicity of the aryl selenone leaving group. The latter reaction is suggested by the present inventors to represent an example of a general approach to synthesis of alkyl aryl selenones.
An additional reaction of selenones is that higher homologs of PhSeO.sub.2 R compounds (where R is aliphatic) at room temperature can undergo .beta.-phenyl group migration reactions, probably through a carbocation mechanism, with a decrease in R-group ring size, acetal formation, among other reactions (Bergman, J., Engman, L., and Siden, J. In: The Chemistry of Organic Selenium and Tellurium Compounds. Vol. 1. (S. Patai and Z. Rappoport, eds.), John Wiley and Sons, N.Y., 1986, pp. 546-558.; Paulmier, C. Selenium Reagents and Intermediates in Organic Synthesis. Pergamon Press, N.Y., 1986, pp. 153-161.; Krief, A., Dumont, W., and De Mahieu, A. F. Tetrahed. Lett. 29:3269-3272, 1988).
The several types of leaving group behavior of the selenonyl group, together with the prior absence of any quantitative kinetic data, therefore, would not allow lead one skilled in the art to expect that 2-chloroethyl aryl selenones would be useful as alkylating agents analogous to clinical ethyleneimines and nitrosoureas.
In a preliminary report, in abstract form, (Kang, S. I., and Spears, C. P. Proc. Am. Assoc. Cancer Res. 30:459, 1989) the inventors described the NBP alkylating activities, reactivities, and in vitro cytotoxicities of several 2-chloroethyl aryl selenones against L1210/0, CCRF-CEM/0, SK-MES-1 and SK-LU-2 cells. This data did not include any drug-resistant cell lines nor was there any disclosure regarding the avoidance of drug resistance.